This project aims to investigate the molecular mechanism by which PTEN is transcriptionally repressed in human breast cancer. PTEN is mutated in roughly 40% of breast cancers, but even in the absence of mutations, PTEN is often transcriptionally repressed, leading to aberrant PI3K pathway activity and subsequent uncontrolled cell growth. In aim 1, the transcriptional profile of PTEN will be defined in normal mammary epithelium; a critical first step for understanding the transcriptional repression of PTEN observed in cancer. To accomplish this goal, normal mammary epithelium will be purified and RNA-seq will be employed to catalog distinct RNA species originating from PTEN and pseudogene PTENP1. Relative abundance will be measured using quantitative real-time PCR (qRT-PCR). The relationship between each of the transcripts identified, and their association with PTEN protein expression will be investigated. Moreover, the difference in PTEN transcriptional profile between the various subtypes of mammary epithelial cells will be determined, as this may be important for the mechanism by which PTEN deficiency contributes to breast cancer. In aim 2, we will identify (1) cancer-specific changes in PTEN transcription and (2) the hypothesized chromatin-based mechanism by which these changes occur. By comparing the results of the RNA-seq and qRT-PCR experiments in normal epithelium to breast cancer biopsies and breast cancer cell lines, changes in the PTEN and PTENP1 transcriptional profile that are specifically associated with breast cancer will be determined. Noncoding RNAs will be investigated as a molecular cause of PTEN repression through the recruitment of repressive complexes to PTEN or by direct action. microRNAs may cause degradation of the nascent PTEN mRNA by binding to seed matches in the 3'UTR; therefore, this will be investigated as an alternative mode of downregulation of PTEN transcript. To implicate candidate repressor complexes in PTEN repression, pharmacological inhibition, chromatin immunoprecipitation (ChIP), and transient knockdowns of identified candidates will be employed. Given that the PTEN/PI3K pathway is dysregulated in many cancers among other diseases, understanding the detailed regulations of PTEN may illuminate a paradigm for diseases characterized by PTEN deficiency and open the door for novel diagnostic and/or therapeutic approaches.